Border-associated macrophages mediate the neuroinflammatory response in an alpha-synuclein model of Parkinson disease

Dopaminergic cell loss due to the accumulation of α-syn is a core feature of the pathogenesis of Parkinson disease. Neuroinflammation specifically induced by α-synuclein has been shown to exacerbate neurodegeneration, yet the role of central nervous system (CNS) resident macrophages in this process remains unclear. We found that a specific subset of CNS resident macrophages, border-associated macrophages (BAMs), play an essential role in mediating α-synuclein related neuroinflammation due to their unique role as the antigen presenting cells necessary to initiate a CD4 T cell response whereas the loss of MHCII antigen presentation on microglia had no effect on neuroinflammation. Furthermore, α-synuclein expression led to an expansion in border-associated macrophage numbers and a unique damage-associated activation state. Through a combinatorial approach of single-cell RNA sequencing and depletion experiments, we found that border-associated macrophages played an essential role in immune cell recruitment, infiltration, and antigen presentation. Furthermore, border-associated macrophages were identified in post-mortem PD brain in close proximity to T cells. These results point to a role for border-associated macrophages in mediating the pathogenesis of Parkinson disease through their role in the orchestration of the α-synuclein-mediated neuroinflammatory response.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Reporting for specific materials, systems and methods ZENODO.7352291, [47] doi:10.5281/ZENODO.7349256, [48] doi:10.5281/ZENODO.7349228, [49]. Single cell RNA sequencing data is available publicly from the NCBI's Gene Expression Omnibus and are accessible at the following link and through the GEO accession number GSE178498 https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE178498. The composite and analyzed data generated in this study are also provided in the Source Data file.
Sex, not gender, was tracked and reported in our experiment. This information was gathered as a part of the patient medical history for postmortem tissue. We did not analyze sex-specific differences, as group numbers were not sufficient for sexbased statistical analyses. However, the Source Data file contains this information and demonstrates that there were no obvious differences between the samples from tissue between sexes.
We did not track/analyze race or ethnicity in the current study.
Age, sex, PD diagnosis status, and additional medical diagnoses were tracked for all human samples. Additional diagnoses are not shared to limit indirect identifiers for patients.  Van Hove et al. 2019) to determine the number of brains to pool per sample in scRNA sequencing steps. No statistical tests were used to determine sample size for analysis of immunostained brains, however, these experiments were meant to corroborate more quantitative flow cytometry findings, and therefore, similar sample sizes were used.
No data were excluded from analyses.
Flow cytometry based quantification of recombination efficiency in CX3CR1 Cre based mice was replicated twice with the same results. T cell quantification in these mice was also replicated twice, yielding the same results. Stereological quantification of neurodegeneration in CX3CR1 Cre based mice was performed once and results are displayed in Figure 1. RNA sequencing experiments were performed once. Flow cytometry of AAV2-GFP and AAV2-SYN transduced mice (quantifying all microglial and BAM markers of activation and T cell/monocyte infiltration) were performed three times resulting in the same outcome. Fate map experiments using TMEM119 Cre based mice were replicated twice with the same results. Interferon gamma based stimulation of TMEM119 Cre based mice was performed once. Flow cytometric quantification of inflammation in TMEM119 Cre based mice was performed once. Immunofluorescence based confirmation of scRNA sequencing findings in AAV2-SYN mice was performed once. Clodronate liposome experiments were performed twice with the same results.
Organisms were allocated to groups in order to provide age and sex-matched controls between groups. Human samples were not randomized, as knowledge of diagnosis was required in order to age and sex match groups.
Blinding was used during stereology analysis, in order to prevent bias in cell counts. Blinding was not used for flow cytometric experiments so that gates capturing markers of inflammation were accurate. Additionally, blinding of these samples was impossible, as it is easily determined which samples are inflamed. Investigators were not blinded during scRNA sequencing analysis, as this analysis was unbiased by nature. All image analyses using ImageJ were performed with the analyzer blinded to group.
For immunohistochemistry, antibodies were validated with no primary controls and by using co-labelling in both inflamed and noninflamed brains or spleens to ensure the labeling of expected cell types. Anti-mouse CD4 and CD8 were validated in spleens, where these cells are abundant. Anti-mouse IBA1, anti-CD68, and anti-MHCII were validated in naive and inflamed brain to ensure the expected morphological changes in microglia were detected. Anti-tyrosine hydroxylase was validated in the ventral midbrain and using anti-NeuN as a co-label. Anti-GPNMB and anti-Apoe were validated with a no-primary control. Anti-pser129 was validated using AAV2-SYN injected brains and pre-formed fibril injected brains as a positive control and naive brains as a negative control. Anti-CD206 was validated using co-labeling with laminin (to label the vasculature) and by labeling whole mount meninges, where CD206+ cells are abundant. Antibodies used in flow cytometry were tested in both brain and spleen and validated in inflamed (stimulated with lipopolysaccharide) and naive brains. Fluorescence minus one controls were used to determine autofluorescence and colabelling was used to determine specificity of the antibody for the expected cell type and inflammatory status. Specifically, anti-CD45, MHCII, CD4, CD8a, interferon gamma, IL-17a, IL-4, IL-10, PD-L1, CD80, CD38, and Ki67 were validated in a naive mouse spleen stimulated with PMA and ionomycin, as these markers are abundant and present in the spleen. Anti-CD11b, CX3CR1, and Ly6C were validated using mouse blood. Identification of cell types that express these markers were consistent with the reported population sizes. Anti-CD68 and MHCII were validated in brain stimulated with lipopolysaccharide to ensure upregulation of inflammatory markers. Antibodies used in postmortem human brain tissue (anti-human CD3, CD4, CD8, and CD68) have been regularly used by the CUMC Department of Pathology and Cell Biology Immuno-stain Lab and were independently validated.